We are studying a regulatory system Saccharomyces cerevisiae, known as general amino acid control, which couples the rate of transcription of a large number of unlinked amino acid biosynthetic genes to amino acid availability. We have shown previously that expression of GCN4, the direct positive effector in this system, is controlled by amino acid availability. This regulation of GCN4 operates at the translation level and is mediated by cis-acting sequences in the 5' end of GNC4 mRNA. CGN4 expression is also controlled by several trans-acting factors, some of which promote GCN4 expression in starvation conditions (GCN) and others which are required for repression in non-starvation conditions (GCD). In the past year, we have completed our analysis of a large number of point mutations which remove the AUG initiation codons of four small open-reading frames located in the 5' leader of GCN4 mRNA. Our results show that these open-reading-frames are critical for the translational control of GCN4, that the individual open-reading-frames have distinct functions, and that interactions between the open-reading-frames are essential for a proper regulatory response. Furthermore, it appears that the GCN and GCD factors regulated GCN4 by controlling the interactions between the upstream open-reading-frames. We have isolated many new good regulatory mutations in five unlinked genes and find that all of these new mutations impair the translational regulation of GCN4. Interestingly, many of the good mutations are conditional lethals. In particular, mutations in GCD1 and GCD12 lead to arrest in the G1 period of the cell cycle at 36 degrees. The gcd1 and gcd2 mutations also show an unexpected allele-specific interaction with previously isolated mutations in the positive regulatory gene GCN3, suggesting that the products of GCD1, GCD12 and GCN3 interact to affect both translational control of GCN4 and an essential function needed for entry into the cell cycle. We have completed the DNA sequence of GCN3 and have begun the preparation of GCN3-specific antisera to be used to examine the cellular location of GCN3 and to probe for interactions between GCN3 and other regulatory proteins.